Polypeptides are increasingly being used as medicaments for the treatment of diseases within all major therapy areas. Treatment of diabetes by chronic insulin administration has been practised for more than 80 years, and therapeutic applications of polypeptides within growth disorders and cancer also have been practised for many years.
Economical processes for the large scale production of polypeptides with a purity sufficiently high for therapeutic applications are crucial for further polypeptide-based therapies to reach the mass market and for the existing therapies to become more widely used.
Polypeptides for therapeutic applications are to be highly purified in order to be efficacious and in order to provide certainty for not causing adverse events upon administration to patients. A number of processing steps used in the synthesis and purification of polypeptides are known to cause racemization of one or more amino acid residues in the target polypeptide. Typically, these conditions are pH extremes and high temperatures, e.g. pH values at above pH 12 (Senderoff et al. J. Pharm. Sci. 87, 183-189 (1998)). The polypeptide variants having a racemized amino acid residue are amenable to separation and identification by state of the art analytical techniques. These variants are undesirable in polypeptides for therapeutic use due to toxicity concerns and because they may have different activity than the desired polypeptide. However, it is a serious challenge to separate these closely related polypeptides in preparative scale, i.e. during industrial manufacture.
Purification of a polypeptide from a mixture is a steps which is normally used several times during the overall manufacturing process for a therapeutic polypeptide. Ion exchange chromatography is often applied in the early and crude separation steps, whereas reverse phase high pressure liquid chromatography (RP-HPLC) is the preferred method for industrial high resolution separation of related polypeptides in the final purification steps. RP-HPLC has proven versatile for the large scale purification of many polypeptides but the process is relatively expensive and has limited capacity.
We have surprisingly found an ion exchange process which can separate the two polypeptide variant the result when racemization of an amino acid residue has taken place. The method is amenable for large scale operation and provides an economical purification step with high capacity.